This proposal seeks funding to develop and characterize measurements that are likely to reflect important aspects of biotin nutrition and to apply these measurements to a rat model of biotin deficiency. The long term goal is estimation of the biotin requirement for man and the prevention of biotin defciency. These investigations are particularly timely and relevant in view of the emerging clinical problems of biotin deficiency on parenteral nutrition (a common nutritional therapy) and biotin-responsive inborn errors of metabolism and in view of questions raised about the importance of biotin in multivitamin supplementation for the general population, in sudden infant death syndrome, and in teratogenesis. These investigations are necessary because very little is known about biotin nutrition in man and methodologic problems cast doubt on much of what is known. These assays include an assay for free and total biotin in serum, urine, and tissue samples, assays for the four biotin-dependent carboxylases in pertinent tissues, a gas chromatography-mass spectrometry assay to quantitate the urinary organic acids that are indices of deficency of two biotin-dependent carboxylases, and an HPLC assay for that will be used to assess catabolites of biotin and to determine which metabolites are detected as biotin in our biotin assay. We have developed a new biotin assay that has advantages over the other assays available. After development, these assays will be applied to an animal model of biotin deficency in order to determine the validity of biotin levels and organic aciduria as indices of acute and chronic biotin deficiency as directly determined by invasive methods that cannot be used in man (e.g. tissue biotin and enzyme activity levels, severity of signs of biotin deficiency). Biotin-deficient animals and isolated perfused livers from these animals will be used to investigate the metabolic repercussions of biotin deficiency in order to better understand the pathogenesis of biotin deficiency and in order to test hypotheses concerning the role of biotin in sudden infant death syndrome.